Pharmacological actions of philanthotoxin analogues on α4β2 and α3β4 nicotinic acetylcholine receptors

Hamid Kachel1,4, Henrik Franzyk2, Kristian Strømgaard2, Denis B. Tikhonov3, Ian R. Mellor1. 1School of Life Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK, 2Department of Molecular Drug Research, University of Copenhagen, Copenhagen, DK-2100, Denmark, 3Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia, 4School of Life Science, University of Zakho, Duhok, Kurdistan Region, Iraq

Neuronal nicotinic acetylcholine receptors (nAChRs) are excitatory ion channels, which have wide subtype diversity due to the many possible subunit combinations, some heteromeric, others homomeric. Each subunit is composed of a large N-terminal segment containing the ACh binding site and four membrane-spanning segments named M1-M4. The pore lumen is lined mainly by the second of these membrane segments, M2. nAChRs play a critical role in many physiological and pathophysiological processes, thus, neuronal nAChRs have become targets for drug discovery and research (Romanelli et al., 2007; Unwin, 2013).

The venom of the Egyptian digger wasp contains a toxin, philanthotoxin-433 (PhTX-433), that works as a strong non-competitive inhibitor of ionotropic glutamate receptors and nAChRs in their target prey (Strømgaard et al., 2000). PhTX-433 has a polyamine tail and aromatic head group. The main obstacle facing the use of the natural toxin (PhTX-433) as a candidate for drug development and understanding pharmacological characteristics of ionotropic receptors is the lack of subtype selectivity. The present study aims to investigate the activity and selectivity of twenty one synthetic analogues of PhTX-343 on rat neuronal α4β2 and α3β4 nAChRs expressed in Xenopus oocytes. We showed that the presence of positive charge in the polyamine tail of PhTX compounds is essential for nAChR subtype selectivity and their removal makes the molecule lose its selectivity. In addition, we identified the key regions and substitutions responsible for increasing PhTX-343 activity, cyclohexylalanine (IC50 of 1 nM on α4β2 and 2 nM on α3β4), and selectivity, phenolic group (30 fold selectivity for α3β4 over α4β2). Analogues having cyclohexylalanine and a phenolic group in the head region showed IC50 values in the low nano-molar and pico-molar (160-400 pM) range. These data suggest that PhTXs could serve as lead compounds for highly potent and selective inhibitors of N-nAChRs.

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